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71.
Nagae M Tsuchiya A Katayama T Yamamoto K Wakatsuki S Kato R 《The Journal of biological chemistry》2007,282(25):18497-18509
1,2-alpha-L-fucosidase (AfcA), which hydrolyzes the glycosidic linkage of Fucalpha1-2Gal via an inverting mechanism, was recently isolated from Bifidobacterium bifidum and classified as the first member of the novel glycoside hydrolase family 95. To better understand the molecular mechanism of this enzyme, we determined the x-ray crystal structures of the AfcA catalytic (Fuc) domain in unliganded and complexed forms with deoxyfuconojirimycin (inhibitor), 2'-fucosyllactose (substrate), and L-fucose and lactose (products) at 1.12-2.10 A resolution. The AfcA Fuc domain is composed of four regions, an N-terminal beta region, a helical linker, an (alpha/alpha)6 helical barrel domain, and a C-terminal beta region, and this arrangement is similar to bacterial phosphorylases. In the complex structures, the ligands were buried in the central cavity of the helical barrel domain. Structural analyses in combination with mutational experiments revealed that the highly conserved Glu566 probably acts as a general acid catalyst. However, no carboxylic acid residue is found at the appropriate position for a general base catalyst. Instead, a water molecule stabilized by Asn423 in the substrate-bound complex is suitably located to perform a nucleophilic attack on the C1 atom of L-fucose moiety in 2'-fucosyllactose, and its location is nearly identical near the O1 atom of beta-L-fucose in the products-bound complex. Based on these data, we propose and discuss a novel catalytic reaction mechanism of AfcA. 相似文献
72.
This protocol details a method for monitoring glucose uptake into single, living mammalian cells using a fluorescent D-glucose derivative, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG), as a tracer. The specifically designed chamber and superfusion system for evaluating 2-NBDG uptake into cells in real time can be combined with other fluorescent methods such as Ca2+ imaging and the subsequent immunofluorescent classification of cells exhibiting divergent 2-NBDG uptake. The whole protocol, including immunocytochemistry, can be completed within 2 d (except for cell culture). The procedure for 2-NBDG synthesis is also presented. 相似文献
73.
Kiyosaki T Matsumoto I Asakura T Funaki J Kuroda M Misaka T Arai S Abe K 《The FEBS journal》2007,274(8):1908-1917
We cloned a new cysteine proteinase of wheat seed origin, which hydrolyzed the storage protein gliadin almost specifically, and was named gliadain. Gliadain mRNA was expressed 1 day after the start of seed imbibition, and showed a gradual increase thereafter. Gliadain expression was suppressed when uniconazol, a gibberellin synthesis inhibitor, was added to germinating seeds. Histochemical detection with anti-gliadain serum indicated that gliadain was present in the aleurone layer and also that its expression intensity increased in sites nearer the embryo. The enzymological characteristics of gliadain were investigated using recombinant glutathione S-transferase (GST)-progliadain fusion protein produced in Escherichia coli. The GST-progliadain almost specifically digested gliadin into low molecular mass peptides. These results indicate that gliadain is produced via gibberellin-mediated gene activation in aleurone cells and secreted into the endosperm to digest its storage proteins. Enzymologically, the GST-progliadain hydrolyzed benzyloxycarbonyl-Phe-Arg-7-amino-4-methylcoumarin (Z-Phe-Arg-NH(2)-Mec) at K(m) = 9.5 microm, which is equivalent to the K(m) value for hydrolysis of this substrate by cathepsin L. Hydrolysis was inhibited by two wheat cystatins, WC1 and WC4, with IC(50) values of 1.7 x 10(-8) and 5.0 x 10(-8) m, respectively. These values are comparable with those found for GST-progliadain inhibition by E-64 and egg-white cystatin, and are consistent with the possibility that, in germinating wheat seeds, gliadain is under the control of intrinsic cystatins. 相似文献
74.
Ando Shiori Funato Michinori Ohuchi Kazuki Inagaki Satoshi Sato Arisu Seki Junko Kawase Chizuru Saito Toshio Nishio Hisahide Nakamura Shinsuke Shimazawa Masamitsu Kaneko Hideo Hara Hideaki 《Neurochemical research》2019,44(7):1773-1779
Neurochemical Research - Spinal muscular atrophy (SMA) is an inherited disease characterized by progressive motor neuron death and subsequent muscle weakness and is caused by deletion or mutation... 相似文献
75.
76.
Ichikawa K Inagaki T Kachi-Tonai H Kojima Y Nakamura T Nishida H Ueno Y Binding P Gabel CA Lucas V McNiff PA Kojima N 《Biochemical and biophysical research communications》2001,286(4):697-700
LL-Z1271alpha, a fungal metabolite, dose-dependently inhibited interleukin-1beta (IL-1beta) production in lipopolysaccharide (LPS)-stimulated human whole blood. Oral administration of LL-Z1271alpha to LPS-challenged mice caused significant lowering in the IL-1beta levels in peritoneal cavity. Data presented suggest that LL-Z1271alpha inhibits IL-1beta production by a novel mechanism as the inhibitory activity was not due to effects on caspase-1 (IL-1beta converting enzyme), the ATP-induced release mechanism or a lysosomotrophic effect. 相似文献
77.
Yagyu K Kitagawa K Irie T Wu B Zeng XT Hattori N Inagaki C 《Journal of neurochemistry》2001,78(3):569-576
Cl(-)-ATPase in the CNS is a candidate for an outwardly directed neuronal Cl(-) transporter requiring phosphatidylinositol-4-phosphate (PI4P) for its optimal activity. To test its pathophysiological changes in a phosphatidylinositol (PI) metabolism disorder, the effects of neurotoxic factors in Alzheimer's disease (AD), amyloid beta proteins (Abetas), on the Cl(-)-ATPase activity were examined using primary cultured rat hippocampal neurons. Amyloid beta proteins (1-40, 1-42 and 25-35) concentration-dependently (1-100 nM) and time-dependently (from 1 h to 6 day) decreased Cl(-)-ATPase activity and elevated intracellular Cl(-) concentrations ([Cl(-)]i), Abeta25-35 being the most potent. Addition of inositol or 8-Br-cyclic GMP completely reversed these Abeta-induced changes. The recoveries in enzyme activity were attenuated by an inhibitor of PI 4-kinase, 10 microM wortmannin or 20 microM quercetin, but not by a PI 3-kinase inhibitor, 50 nM wortmannin or 10 microM LY294002. The PI, PIP and PIP2 levels of the plasma membrane-rich fraction were lower in the Abeta-treated cells as compared with each control. In the Abeta-exposed culture, but not in control, stimulation by 10 microM glutamate for 10 min significantly increased fragmentation of DNA and decreased cell viability. Addition of inositol or 8-Br-cyclic GMP prevented the effect of Abeta-treatment on the neurotoxicity of glutamate. Thus, Abetas reduce neuronal Cl(-)-ATPase activity, resulting in an increase in [Cl(-)]i probably by lowering PI4P levels, and this may reflect a pre-apoptotic condition in early pathophysiological profiles of AD. 相似文献
78.
Genes encoding the vacuolar Na+/H+ exchanger and flower coloration 总被引:22,自引:0,他引:22
Yamaguchi T Fukada-Tanaka S Inagaki Y Saito N Yonekura-Sakakibara K Tanaka Y Kusumi T Iida S 《Plant & cell physiology》2001,42(5):451-461
Vacuolar pH plays an important role in flower coloration: an increase in the vacuolar pH causes blueing of flower color. In the Japanese morning glory (Ipomoea nil or Pharbitis nil), a shift from reddish-purple buds to blue open flowers correlates with an increase in the vacuolar pH. We describe details of the characterization of a mutant that carries a recessive mutation in the Purple (Pr) gene encoding a vacuolar Na+/H+ exchanger termed InNHX1. The genome of I. nil carries one copy of the Pr (or InNHX1) gene and its pseudogene, and it showed functional complementation to the yeast nhx1 mutation. The mutant of I. nil, called purple (pr), showed a partial increase in the vacuolar pH during flower-opening and its reddish-purple buds change into purple open flowers. The vacuolar pH in the purple open flowers of the mutant was significantly lower than that in the blue open flowers. The InNHX1 gene is most abundantly expressed in the petals at around 12 h before flower-opening, accompanying the increase in the vacuolar pH for the blue flower coloration. No such massive expression was observed in the petunia flowers. Since the NHX1 genes that promote the transport of Na+ into the vacuoles have been regarded to be involved in salt tolerance by accumulating Na+ in the vacuoles, we can add a new biological role for blue flower coloration in the Japanese morning glory by the vacuolar alkalization. 相似文献
79.
Ogura K Nagata K Hatanaka H Habuchi H Kimata K Tate S Ravera MW Jaye M Schlessinger J Inagaki F 《Journal of biomolecular NMR》1999,13(1):11-24
Fibroblast growth factors (FGFs) bind to extracellular matrices, especially heparin-like carbohydrates of heparansulfate proteoglycans which stabilize FGFs to protect against inactivation by heat, acid, proteolysis and oxidation. Moreover, binding of FGFs to cell surface proteoglycans promotes to form oligomers, which is essential for receptor oligomerization and activation. In the present study, we determined the solution structure of acidic FGF using a series of triple resonance multi-dimensional NMR experiments and simulated annealing calculations. Furthermore, we prepared the sample complexed with a heparin-derived hexasaccharide which is a minimum unit for aFGF binding. From the chemical shift differences between free aFGF and aFGF-heparin complex, we concluded that the major heparin binding site was located on the regions 110–131 and 17–21. The binding sites are quite similar to those observed for bFGF-heparin hexasaccharide complex, showing that both FGFs recognize heparin- oligosaccharides in a similar manner. 相似文献
80.
Molecular genetics of dopa-responsive dystonia 总被引:4,自引:0,他引:4
The causative genes of two types of hereditary dopa-responsive dystonia (DRD) due to dopamine (DA) deficiency in the nigrostriatum DA neurons have been elucidated. Autosomal dominant DRD (AD-DRD) was originally described by Segawa as hereditary progressive dystonia with marked diurnal fluctuation (HPD). We cloned the human GTP cyclohydrolase I (GCH1) gene, and mapped the gene to chromosome 14q22.1-q22.2 within the HPD/DRD locus, which had been identified by linkage analysis. GCH1 isthe rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin (BH4), the cofactor for tyrosine hydroxylase (TH), which is the first and rate-limiting enzyme of DA synthesis. We proved that the GCH1 gene is the causative gene for HPD/DRD based on the identification of mutations of the gene in the patients and decreases in the enzyme activity expressed in mononuclear blood cells to 2-20% of the normal value. About 60 different mutations (missense, nonsense, and frameshift mutations) in the coding region or in the exon-intron junctions of the GCH1 gene have been reported in patients with AD-DRD all over the world. Recent findings indicate that the decreased GCH1 activity in AD-DRD may be caused by the negative interaction of the mutated subunit with the wild-type one, i.e., a dominant negative effect, and/or by decreases in the levels of GCH1 mRNA and protein caused by inactivation of one allele of the GCH1 gene. Autosomal recessive DRD (AR-DRD) with Segawa's syndrome was discovered in Germany. The AR-DRD locus was mapped to chromosome 11p15.5 in the chromosomal site of the TH gene. In the AR-DRD with Segawa's syndrome, a point mutation in TH (Gln381Lys) resulted in a pronounced decrease in TH activity to about 15% of that of the wild type. Several missense mutations in the TH gene have been found in AR-DRD in Europe. The phenotype of AR-DRD with the Leu205Pro mutation in the TH gene, which produces a severe decrease in TH activity to 1.5% of that of the wild type, was severe, not dystonia/Segawa's syndrome, but early-onset parkinsonism. However, a marked improvement of all clinical symptoms with a low dose of L-dopa was reported in AR-DRD/parkinsonism patients. These findings on DRD indicate that the nigrostriatal DA neurons may be most susceptible to the decreases in GCH1 activity, BH4 level, TH activity, and DA level, and that DRD is the DA deficiency without neuronal death in contrast to juvenile parkinsonism or Parkinson's disease with DA cell death. 相似文献